Recent studies on lipids have revealed that highly unsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) have various functions such as the improvement of learning function, the prevention of arteriosclerosis, and the improvement function of lipid metabolism. In particular, it has been revealed that the intake of DHA in a form bonded to a phospholipid such as phosphatidylcholine provides higher antioxidant activity and higher stability than those of the triglyceride form as well as it leads to good absorption to readily provide physiological activities of the DHA. Functional fatty acids other than DHA, such as EPA, conjugated linoleic acid, and arachidonic acid are also expected to achieve higher physiological activities by the bonding to a phospholipid.
Methods for producing a phospholipid bonded with a functional fatty acid such as DHA are classified into a method of extraction of a natural product and a method of synthesis from a material such as soybean phospholipids. Specific examples of the former method include a method of extraction of a DHA-bonded phospholipid from aquatic animal roes as a material (Patent Document 1) and a method of extraction from marine products such as a squid with an organic solvent (Patent Document 2). However, these methods cannot produce phospholipids bonded with functional fatty acids other than DHA because the materials are expensive and cannot stably be supplied and the composition of phospholipid depends on materials.
Examples of methods capable of introducing a desired fatty acid not depending on a material composition include a method by adding any fatty acid to a culture solution of a microorganism to produce a phospholipid bonded with the fatty acid by the microorganism (Patent Document 3). However, the method produces the phospholipid in a small amount from a large amount of the culture solution and thus the production efficiency is poor.
Among the latter methods, examples of the method of bonding DHA to soybean phospholipids and the like include a method of adding a high-permittivity substance capable of forming hydrogen bonds to a reaction system of lipase and phospholipase (Patent Document 4). However, the method can achieve a high reaction rate in the reaction of a lysophospholipid and a fatty acid by the lipase but cannot achieve a high reaction rate by phospholipase A2. Furthermore, it is important for the expression of physiological activities of the DHA-bonded phospholipid that DHA is bonded to the 2-position, but a target fatty acid is mainly bonded to the 1-position in a phospholipid through a reaction by the lipase, and therefore such a method is not highly practical.
Meanwhile, as a method for efficiently bonding a desired fatty acid to the 2-position in a phospholipid, there have been reported some bonding methods using phospholipase A2 in glycerol (Patent Document 5 and Non-patent Document 1). However in these reports, toxic chloroform-methanol is used for extraction after the reaction. Thus, the solvent cannot be used depending on an intended use of the phospholipid, or an apparatus for removing the solvent is required. Moreover, the phospholipase A2 is expensive, and hence such a method is required to reduce costs.
In a common enzyme reaction, enzyme immobilization is widely performed for the efficient use of the enzyme. However, there have been reports that when an immobilized phospholipase A2 is used in esterification by the phospholipase A2, the esterification is unlikely to efficiently proceed even when a fatty acid is used in a large amount with respect to a lysophospholipid (Non-patent Document 2 and Non-patent Document 3).